A deep-sea pathogenic Bacillus subtilis isolate employs different strategies to escape the killing of teleost and murine complements.

Bacillus subtilis subsp. subtilis G7 was isolated from a deep-sea hydrothermal vent and is pathogenic to pathogenic to fish (Japanese flounder) and mice. G7 is able to survive in host sera and phagocytes. In this study, we investigated the underlying mechanism of G7 serum resistance. We found that (i) the remaining complement activity was very low in G7-incubated flounder serum but high in G7-incubated mouse serum; (ii) cleaved C3 and C5 components were detected on flounder serum-incubated G7 but not on mouse serum-incubated G7; (iii) abundant uncleaved C5 was localized in G7-incubated mouse, but not flounder, serum; (iv) G7-incubated flounder, but not mouse, serum exhibited strong chemotactic activity; (v) pre-treatment with low-dose lysozyme abolished the serum resistance of G7. Hence, G7 activates flounder complement but is protected from complement-mediated destruction by its cell wall structure, while G7 prevents the activation of mouse complement. These results indicate that G7 employs different mechanisms to avoid the complement killing of different hosts.

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis.

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.

Nosocomial bacteremia and catheter infection by Bacillus cereus in an immunocompetent patient.

We present a case of Bacillus cereus bacteremia and catheter infection in an immunocompetent patient subjected to abdominal surgery, who recovered following central catheter removal and treatment with piperacillin/tazobactam.

Bacillus cereus necrotizing cellulitis mimicking clostridial myonecrosis: case report and review of the literature.

We describe a case of rapidly progressive necrotizing cellulitis in an immunocompromised farmer caused by Bacillus cereus, and review 15 additional cases of serious soft tissue infection due to this organism reported in the English language literature. These cases illustrate the potential for B. cereus to cause fulminant soft tissue disease indistinguishable from that caused by clostridia.

In vitro model of adhesion and invasion by Bacillus piliformis.

An in vitro model of Bacillus piliformis infection was developed to investigate the mechanisms of adhesion and internalization of this obligate intracellular bacterium. Adhesion and internalization events were examined by electron microscopic evaluation of infected Caco-2 cell monolayers. A few bacteria were identified in apical surface invaginations and in vacuoles subjacent to the apical surface, whereas the majority of bacteria were observed free within the cytoplasm, suggesting that B. piliformis entered epithelial cells via a phagocytic process and rapidly escaped the phagosome. To confirm that host cell phagocytosis was involved in entry of B. piliformis into mammalian cells, Intestine 407 cells were treated with the phagocytic inhibitor cytochalasin D, infected with B. piliformis, and evaluated for bacterial internalization by double-fluorescence labeling. The results showed decreased intracellular bacteria, suggesting that internalization was dependent on host cell microfilament function. To examine the role of B. piliformis in internalization, growth of live and Formalin-killed bacteria was compared. Dead bacteria were not internalized, suggesting that B. piliformis actively participates in internalization. B. piliformis appears to enter host cells by a bacterially directed phagocytic process. The in vitro system described should prove invaluable in further investigations of B. piliformis pathogenic mechanisms.

Bacillus cereus pneumonia in a patient with acute lymphoblastic leukemia.

Reported here is a case of Bacillus cereus pneumonia that occurred in a patient with acute lymphoblastic leukemia. The presentation was severe, essentially marked by respiratory distress and pleuritic chest pain. Classic empirical treatment initiated for febrile neutropenia did not cover this rare pathogen and appropriate therapy was therefore delayed. B. cereus is most often a culture contaminant, but it can also be responsible for self-limited gastrointestinal intoxication and, more rarely, severe systemic diseases. Virulence in the case of systemic disease is attributed to tissue necrosis mediated by toxin release. B. cereus pneumonia, as described in the English-language literature, mainly affects immunocompromised patients and most often has a fatal outcome. Thus, the identification of B. cereus in clinical specimens of severely ill immunocompromised patients should lead physicians to question its clinical significance.

Ontogeny and behaviour of early macrophages in the zebrafish embryo.

In the zebrafish embryo, the only known site of hemopoieisis is an intra-embryonic blood island at the junction between trunk and tail that gives rise to erythroid cells. Using video-enhanced differential interference contrast microscopy, as well as in-situ hybridization for the expression of two new hemopoietic marker genes, draculin and leucocyte-specific plastin, we show that macrophages appear in the embryo at least as early as erythroid cells, but originate from ventro-lateral mesoderm situated at the other end of the embryo, just anterior to the cardiac field. These macrophage precursors migrate to the yolksac, and differentiate. From the yolksac, many invade the mesenchyme of the head, while others join the blood circulation. Apart from phagocytosing apoptotic corpses, these macrophages were observed to engulf and destroy large amounts of bacteria injected intravenously; the macrophages also sensed the presence of bacteria injected into body cavities that are isolated from the blood, migrated into these cavities and eradicated the microorganisms. Moreover, we observed that although only a fraction of the macrophage population goes to the site of infection, the entire population acquires an activated behaviour, similar to that of activated macrophages in mammals. Our results support the notion that in vertebrate embryos, macrophages endowed with proliferative capacity arise early from the hemopoietic lineage through a non-classical, rapid differentiation pathway, which bypasses the monocytic series that is well-documented in adult hemopoietic organs.